Infection by bacterial pathogens continues to be a major health concern throughout the world. For example, during the past decade the incidence of cholera spread to more areas of the globe than had occurred in the previous 100 years. A better understanding of the molecular bases of pathogenesis may provide new ways to combat bacterial infection. The goal of this research is to discern the mechanisms by which physiological signals in the environment within the human host are converted to molecular interactions that govern the expression of virulence genes of the infecting bacterium. The model system to be analyzed is the Vibrio cholerae ToxR virulence regulon, for which a number of parameters that influence gene expression, as well as many of the regulators and target genes, are known and partially characterized. The target virulence genes include the tcp operon, toxT, and other "ToxR activated" genes present on the Vibrio cholerae TCP pathogenicity island, as well as the ctx operon present on a lysogenic bacteriophage. The regulators are encoded by genes distributed around the genome, including toxRS, aph.4, aphB, hns, and crp, as well as the tcpPH and toxT genes present on the pathogenicity island. It has recently been determined that multiple regulators function at each target gene promoter. The current proposal focuses on a subset of target promoters and regulators to understand how these regulators function in an interaction with growth condition signals, the promoters, and with each other to control gene expression. In addition, the identity of additional target genes for which expression is influenced by the regulatory proteins that are not encoded within the Vibrio TCP pathogenicity island will help to better understand the precise chemical and physical responses that are being converted into virulence gene expression mechanisms. Correlating virulence gene expression together with regulatory responses that modulate bacterial physiology represents a new approach that utilizes knowledge of the genome to further our understanding of the basis of virulence gene regulation. Finally, new approaches to monitor virulence gene expression both in vitro and in vivo will provide a means to correlate these two events and to identify additional genes involved in regulation. These experiments will likely reveal novel virulence factor genes that may prove to be useful vaccine or therapeutic targets. A further understanding of virulence gene expression will also help in the development of ways to modulate it either in vivo or in vitro for improved vaccine production or overproduction of virulence factors for structural analyses.